Project Summary Gonadotropin releasing hormone (GnRH) neurons are the final neural output controlling the secretion of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary and ovarian steroid hormones (estradiol and progesterone) in female mammals. In turn, GnRH secretion is regulated by gonadal steroid hormones via a feedback mechanisms. Throughout the estrous cycle across species, low levels of estradiol inhibit GnRH secretion via negative steroid hormone feedback. High levels of estradiol during the late follicular phase result in positive feedback, leading to the preovulatory GnRH/LH surge. Finally, progesterone, plays an important role in negative feedback during the luteal phase of the cycle. GnRH neurons do not directly respond to gonadal steroid hormone feedback, either negative or positive, as they do not contain the appropriate steroid receptors. It is therefore hypothesized that GnRH neurons rely on an afferent neuronal network to convey this influence. Recent evidence suggests that Kisspeptin, Neurokinin B and Dynorphin (KNDy) neurons in the arcuate nucleus of the hypothalamus play a pivotal role in conveying the feedback effects of gonadal steroid hormones on GnRH secretion during the estrous cycle. The long-term goal of this project is to further understand the mechanisms by which gonadal steroid hormones regulate KNDy neurons using female sheep as the animal model. In the first aim I will use whole tissue clearing approaches and light sheet microscopy to test the hypothesis that specific subpopulations of KNDy neurons are involved in regulating the negative and positive feedback influences of ovarian steroids on pulsatile and surge modes of GnRH/LH secretion, respectively. The second aim will test the hypothesis that KNDy neurons are heterogeneous in their response to gonadal steroids using a sensitive in situ hybridization technique, i.e. RNAscope, that allows for simultaneous visualization and analysis of multiple mRNA transcripts in KNDy neurons. Together, these studies will provide a greater level of knowledge of neuroendocrine substrates responsible for control of reproduction, and may reveal new targets for future therapeutic intervention for treatment of reproductive disorders. Moreover, these studies will provide a unique training experience that will contribute towards my goal to become an independent scientist.